Monitoring coronary blood flow by laser speckle contrast imaging after myocardial ischaemia reperfusion injury in adult and aged mice.

Introduction: Investigating coronary microvascular perfusion responses after myocardial infarction (MI) would aid in the development of flow preserving therapies. Laser speckle contrast imaging (LSCI) is a powerful tool used for real-time, non-contact, full-field imaging of blood flow in various tissues/organs. However, its use in the beating heart has been limited due to motion artifacts. Methods: In this paper, we report the novel use of LSCI, combined with custom speckle analysis software (SpAn), to visualise and quantitate changes in ventricular perfusion in adult and aged mice undergoing ischaemia-reperfusion (IR) injury. The therapeutic benefit of inhibiting the actions of the pro-inflammatory cytokine interleukin-36 (IL-36) was also investigated using an IL-36 receptor antagonist (IL-36Ra). Results: Imaging from uncovered and covered regions of the left ventricle demonstrated that whilst part of the LSCI flux signal was derived from beating motion, a significant contributor to the flux signal came from ventricular microcirculatory blood flow. We show that a biphasic flux profile corresponding to diastolic and systolic phases of the cardiac cycle can be detected without mathematically processing the total flux data to denoise motion artifacts. Furthermore, perfusion responses to ischaemia and postischaemia were strong, reproducible and could easily be detected without the need to subtract motion-related flux signals. LSCI also identified significantly poorer ventricular perfusion in injured aged mice following IR injury which markedly improved with IL-36Ra. Discussion: We therefore propose that LSCI of the heart is possible despite motion artifacts and may facilitate future investigations into the role of the coronary microcirculation in cardiovascular diseases and development of novel therapies.

Improving vasculoprotective effects of MSCs in coronary microvessels - benefits of 3D culture, sub-populations and heparin.

Introduction: Opening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy. Methods: Intravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonally-derived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods. Results: 3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines. Conclusion: This novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BM-derived MSC sub-population could achieve this.

Imaging the injured beating heart intravitally and the vasculoprotection afforded by haematopoietic stem cells.

AIMS: Adequate microcirculatory perfusion, and not just opening of occluded arteries, is critical to salvage heart tissue following myocardial infarction. However, the degree of microvascular perfusion taking place is not known, limited primarily by an inability to directly image coronary microcirculation in a beating heart in vivo. Haematopoietic stem/progenitor cells (HSPCs) offer a potential therapy but little is known about their homing dynamics at a cellular level and whether they protect coronary microvessels. This study used intravital microscopy to image the anaesthetized mouse beating heart microcirculation following stabilization. METHODS AND RESULTS: A 3D-printed stabilizer was attached to the ischaemia-reperfusion injured (IRI) beating heart. The kinetics of neutrophil, platelet and HSPC recruitment, as well as functional capillary density (FCD), was imaged post-reperfusion. Laser speckle contrast imaging (LSCI) was used for the first time to monitor ventricular blood flow in beating hearts. Sustained hyperaemic responses were measured throughout reperfusion, initially indicating adequate flow resumption. Intravital microscopy confirmed large vessel perfusion but demonstrated poor transmission of flow to downstream coronary microvessels. Significant neutrophil adhesion and microthrombus formation occurred within capillaries with the latter occluding them, resulting in patchy perfusion and reduced FCD. Interestingly, 'patrolling' neutrophils were also observed in capillaries. Haematopoietic stem/progenitor cells readily trafficked through the heart but local retention was poor. Despite this, remarkable anti-thromboinflammatory effects were observed, consequently improving microvascular perfusion. CONCLUSION: We present a novel approach for imaging multiple microcirculatory perturbations in the beating heart with LSCI assessment of blood flow. Despite deceptive hyperaemic responses, increased microcirculatory flow heterogeneity was seen, with non-perfused areas interspersed with perfused areas. Microthrombi, rather than neutrophils, appeared to be the major causative factor. We further applied this technique to demonstrate local stem cell presence is not a pre-requisite to confer vasculoprotection. This is the first detailed in vivo characterization of coronary microcirculatory responses post-reperfusion injury.

Pretreatment of Mesenchymal Stem Cells Manipulates Their Vasculoprotective Potential While Not Altering Their Homing Within the Injured Gut.

Mesenchymal stem cells (MSCs) have shown therapeutic promise in many experimental and clinical models of inflammation. However, a commonly reported feature of MSC transplantation is poor homing to injured tissues. Previously, we have shown that pretreatment with cytokines/chemical factors enhances hematopoietic SC adhesion within intestinal microvasculature following ischemia-reperfusion (IR) injury. Using intravital microscopy, the ability of similar pretreatment strategies to enhance the recruitment of murine MSCs to murine intestinal microvasculature following IR injury was investigated. Primary MSCs were isolated from bone marrow and selected on the basis of platelet-derived growth factor receptor-α and SC antigen-1 positivity (PDGFRα(+) /Sca-1(+) ). MSC recruitment was similar in IR injured gut mucosa when compared with sham operated controls, with limited cell adhesion observed. MSCs appeared contorted in microvessels, suggesting physical entrapment. Although not recruited specifically by injury, MSC administration significantly reduced neutrophil recruitment and improved tissue perfusion in the severely injured jejunum. Vasculoprotective effects were not demonstrated in the lesser injured ileum. Pretreatment of MSCs with tumor necrosis factor (TNF)-α, CXCL12, interferon (IFN)-γ, or hydrogen peroxide did not enhance their intestinal recruitment. In fact, TNFα and IFNγ removed the previous therapeutic ability of transplanted MSCs to reduce neutrophil infiltration and improve perfusion in the jejunum. We provide direct evidence that MSCs can rapidly limit leukocyte recruitment and improve tissue perfusion following intestinal IR injury. However, this study also highlights complexities associated with strategies to improve MSC therapeutic efficacy. Future studies using cytokine/chemical pretreatments to enhance MSC recruitment/function require careful consideration and validation to ensure therapeutic function is not impeded.

Mesenchymal stem cell priming: fine-tuning adhesion and function.

There is significant interest in the use of mesenchymal stem cells (MSCs) as a potential therapeutic modality in disease and disorder, particularly those with an inflammation-based component such as coronary, renal and hepatic diseases. While there is no question that MSCs possess the capability to manipulate an ongoing inflammatory injury, the recruitment of these cells to injured sites is generally poor, and thus, open to manipulation. Enhancing the localised recruitment of MSCs to injured tissues may enhance the efficiency and efficacy of this mode of therapy. A number of techniques exist in the literature to improve the recruitment of MSCs to injured tissues, including the use of cytokines, chemical modifications and coating with either synthetic or biological particles. In addition to enhancing MSC recruitment, there is an increasing body of work examining techniques which may enhance the anti-inflammatory activity of these cells. This review will summarise the literature around these topics. This first section of this review summarises the current literature with regard to MSC homing and their recruitment during conditions of injury. In relation to the anti-inflammatory activity of MSCs, the role of systemic versus local activity will be discussed. The second part of the review focuses on the role of pretreatments in MSC therapy and how these may have potential for not only enhancing the recruitment of MSCs, but also their anti-inflammatory capabilities. In summary, it is clear that there is significant potential to improve the efficiency of MSC therapy and the techniques discussed in this review may be central to this in the future.

Modulating the Adhesion of Haematopoietic Stem Cells with Chemokines to Enhance Their Recruitment to the Ischaemically Injured Murine Kidney.

INTRODUCTION: Renal disease affects over 500 million people worldwide and is set to increase as treatment options are predominately supportive. Evidence suggests that exogenous haematopoietic stem cells (HSCs) can be of benefit but due to the rarity and poor homing of these cells, benefits are either minor or transitory. Mechanisms governing HSC recruitment to injured renal microcirculation are poorly understood; therefore this study determined (i) the adhesion molecules responsible for HSC recruitment to the injured kidney, (ii) if cytokine HSC pre-treatment can enhance their homing and (iii) the molecular mechanisms accountable for any enhancement. METHODS: Adherent and free-flowing HSCs were determined in an intravital murine model of renal ischaemia-reperfusion injury. Some HSCs and animals were pre-treated prior to HSC infusion with function blocking antibodies, hyaluronidase or cytokines. Changes in surface expression and clustering of HSC adhesion molecules were determined using flow cytometry and confocal microscopy. HSC adhesion to endothelial counter-ligands (VCAM-1, hyaluronan) was determined using static adhesion assays in vitro. RESULTS: CD49d, CD44, VCAM-1 and hyaluronan governed HSC adhesion to the IR-injured kidney. Both KC and SDF-1α pre-treatment strategies significantly increased HSC adhesion within injured kidney, whilst SDF-1α also increased numbers continuing to circulate. SDF-1α and KC did not increase CD49d or CD44 expression but increased HSC adhesion to VCAM-1 and hyaluronan respectively. SDF-1α increased CD49d surface clustering, as well as HSC deformability. CONCLUSION: Increasing HSC adhesive capacity for its endothelial counter-ligands, potentially through surface clustering, may explain their enhanced renal retention in vivo. Furthermore, increasing HSC deformability through SDF-1α treatment could explain the prolonged systemic circulation; the HSC can therefore continue to survey the damaged tissue instead of becoming entrapped within non-injured sites. Therefore manipulating these mechanisms of HSC recruitment outlined may improve the clinical outcome of cellular therapies for kidney disease.

Mechanisms of adhesion and subsequent actions of a haematopoietic stem cell line, HPC-7, in the injured murine intestinal microcirculation in vivo.

OBJECTIVES: Although haematopoietic stem cells (HSCs) migrate to injured gut, therapeutic success clinically remains poor. This has been partially attributed to limited local HSC recruitment following systemic injection. Identifying site specific adhesive mechanisms underpinning HSC-endothelial interactions may provide important information on how to enhance their recruitment and thus potentially improve therapeutic efficacy. This study determined (i) the integrins and inflammatory cyto/chemokines governing HSC adhesion to injured gut and muscle (ii) whether pre-treating HSCs with these cyto/chemokines enhanced their adhesion and (iii) whether the degree of HSC adhesion influenced their ability to modulate leukocyte recruitment. METHODS: Adhesion of HPC-7, a murine HSC line, to ischaemia-reperfused (IR) injured mouse gut or cremaster muscle was monitored intravitally. Critical adhesion molecules were identified by pre-treating HPC-7 with blocking antibodies to CD18 and CD49d. To identify cyto/chemokines capable of recruiting HPC-7, adhesion was monitored following tissue exposure to TNF-α, IL-1ß or CXCL12. The effects of pre-treating HPC-7 with these cyto/chemokines on surface integrin expression/clustering, adhesion to ICAM-1/VCAM-1 and recruitment in vivo was also investigated. Endogenous leukocyte adhesion following HPC-7 injection was again determined intravitally. RESULTS: IR injury increased HPC-7 adhesion in vivo, with intestinal adhesion dependent upon CD18 and muscle adhesion predominantly relying on CD49d. Only CXCL12 pre-treatment enhanced HPC-7 adhesion within injured gut, likely by increasing CD18 binding to ICAM-1 and/or CD18 surface clustering on HPC-7. Leukocyte adhesion was reduced at 4 hours post-reperfusion, but only when local HPC-7 adhesion was enhanced using CXCL12. CONCLUSION: This data provides evidence that site-specific molecular mechanisms govern HPC-7 adhesion to injured tissue. Importantly, we show that HPC-7 adhesion is a modulatable event in IR injury and further demonstrate that adhesion instigated by injury alone is not sufficient for mediating anti-inflammatory effects. Enhancing local HSC presence may therefore be essential to realising their clinical potential.

Enhancing the adhesion of hematopoietic precursor cell integrins with hydrogen peroxide increases recruitment within murine gut.

Hematopoietic stem cells (HSCs) migrate to injury sites and aid in tissue repair. However, clinical success is poor and is partially due to limited HSC recruitment. We hypothesized that HSC pretreatment with H2O2 would enhance their recruitment to injured gut. As HSCs are rare cells, the number of primary cells obtained from donors is often inadequate for functional experiments. To circumvent this, in this study we utilized a functionally relevant cell line, HPC-7. Anesthetized mice were subjected to intestinal ischemia-reperfusion (IR) injury, and HPC-7 recruitment was examined intravitally. Adhesion to endothelial cells (ECs), injured gut sections, and ICAM-1/VCAM-1 protein were also quantitated in vitro. H2O2 pretreatment significantly enhanced HPC-7 recruitment to injured gut in vivo. A concomitant reduction in pulmonary adhesion was also observed. Enhanced adhesion was also observed in all in vitro models. Increased clustering of α4 and ß2 integrins, F-actin polymerization, and filopodia formation were observed in pretreated HPC-7s. Importantly, H2O2 did not reduce HPC-7 viability or proliferative ability. HPC-7 recruitment to injured gut can be modulated by H2O2 pretreatment. This may be through increasing the affinity or avidity of surface integrins that mediate HPC-7 homing to injured sites or through stimulating the migratory apparatus. Strategies that enhance hematopoietic stem/progenitor cell recruitment may ultimately affect their therapeutic efficacy.